[Quality evaluation of Polygonatum cyrtonema based on HPLC fingerprint and multi-component quantitative analysis].

The medicinal and edible Polygonatum cyrtonema is one of the original species of Polygonati Rhizoma. In this study,HPLC fingerprints for 25 batches of P. cyrtonema from 6 provinces were established. A total of 14 common peaks were identified and the similarities of the fingerprints were in the range of 0. 939-0. 999. In additon, the partial least squares-discriminant analysis(PLSDA) demonstrated that the samples had low discriminability except for JX-1 and most components of them had no significant correlation with environmental factors such as longitude, latitude, and altitude. Thus, chemical composition specificity of P. cyrtonema in natural distribution areas had no obvious regularity and their variation might be induced by the local environment. This conclusion explained the lack of records about Dao-di area of Polygonati Rhizoma. However, JX-1 boasted significantly higher content of 5-hydroxymethylfurfural(HMF) and 4',5,7-trihydroxy-6,8-dimethylhomoisoflavone( HIF), thick and long inflorescence and rhizome, and extremely high yield. Therefore, excellent variety of P. cyrtonema might have great potential to improve the quality and yield of Polygonati Rhizoma. Moreover, three components of HMF, polygonalline A(PA), and HIF were identified in the fingerprint. Among them, HMF has the activities of blood rheology improvement, antioxidation, and anti-myocardial ischemia and PA is an indolizine alkaloid with potential anti-inflammatory activity. HIF, the characteristic homoisoflavone in Polygonatum, has the pharmacological activities of regulating blood glucose and anti-tumor. A quantitative analysis method can provide a theoretical basis for the improvement of the quality evaluation of Polygonati Rhizoma.

Diagnosis of Hyperacute and Acute Ischaemic Stroke: The Potential Utility of Exosomal MicroRNA-21-5p and MicroRNA-30a-5p.

BACKGROUND: Early and accurate diagnosis of ischaemic stroke (IS) requires the use of an optimized biomarker. Exosomal microRNAs have the potential to serve as biomarkers owing to their stability and specificity. We investigated the expression levels of plasma-derived exosomal microRNA-21-5p and microRNA-30a-5p in the different phases of IS. METHODS: One hundred forty-three patients with IS and 24 non-stroke controls were enrolled. The patients were divided into the following 5 groups: 1 group for the hyperacute phase IS (HIS, within 6 h); two for the acute phase IS (AIS, including days 1-3 and days 4-7); one for the subacute phase IS (SIS, days 8-14); and one for the recovery phase IS (RIS, days >14). Plasma exosomes were isolated using a QIAGEN exoRNeasy kit and examined by transmission electron -microscopy, nanoparticle tracking, and flow cytometry. The expression levels of miRNA-21-5p and miRNA-30a-5p were detected by quantitative real-time polymerase chain reaction. RESULTS: The plasma exosomal miR-21-5p levels in SIS and RIS were significantly higher than that in controls (p < 0.05 and p < 0.01 respectively). The levels of miR-30a-5p in HIS were significantly higher (p < 0.05) and in AIS (days 1-3) were lower than that in controls (p < 0.05). In AIS (days 1-3), both miRNAs were decreased compared with the HIS group (p = 0.053 and 0.001, respectively). The area under the curve (AUC) of the miR-21-5p was 0.714 for SIS (95% CI 0.570-0.859, p = 0.007), 0.734 for RIS (95% CI 0.596-0.871, p = 0.003); the AUC of the miR-30a-5p was 0.826 for HIS (95% CI 0.665-0.988, p = 0.001), 0.438 for AIS (days 1-3; 95% CI 0.240-0.635, p = 0.516). CONCLUSIONS: The plasma-derived exosomal miR-21-5p and miRNA-30a-5p in combination are promising biomarkers for diagnosing IS and distinguishing among HIS, SIS, and RIS, especially miRNA-30a-5p for the diagnosis of the HIS phase. Our results provide a new reference for clinicians to apply in early-stage diagnosis and identifies the possible value of biomarkers for IS thrombolysis therapy.

[SCoT differential expression of cold resistance related genes in Dendrobium officinale under low temperature stress].

In order to study the molecule mechanism of the differential expression in Dendrobium officinale under low temperature, the high cold resistance germplasms were used for constructing the RNA pools. SCoT markers were used to analyze the different cDNA pools transcribed from the RNA pools. 11 transcripts derived fragments from 500 cDNA amplified bands were amplified by 64 primers, and were sorted out, cloned, sequenced and analyzed. The results showed that cDNA pools with SCoT markers could be used for differential display in D. officinale under low temperature stress. Sequence analysis indicated that the transcripts derived fragments were significantly homologous in nucleotide sequence with membrane-associated proteins, osmotic regulation protein, CBF transcriptional factor, resistance protein. One left gene segments functions were still unknown, which may be related to the cold resistant gene expression in D. officinale.

[Cloning and expression analysis of HSP70 gene from Dendrobium officinale under low temperature stress].

OBJECTIVE: To investigate HSP70 gene expression from Dendrobium officinale under low temperature stress, which will provide the molecular biological foundation for breeding the low temperature resistant strain. METHOD: HSP70 gene full length cDNA was cloned by rapid amplification of cDNA ends (RACE) on the basis of HSP70 gene fragment sequences, and the structure and function of HSP70 gene were deduced. The expression of HSP70 under low temperature stress was detected by RT-PCR. RESULT: The full length of HSP70 gene cDNA was 2 296 bp containing a 1 944 bp open reading frame (ORF) that encoded a protein of 647 amino acids. Its amino acids sequence had typical HSP70 characteristics and high homology with other plant's HSP70. Cold stress expression analysis showed that expression of the HSP70 gene could be induced by low temperature. CONCLUSION: The HSP70 gene of D. officinale was successfully cloned and reported for the first time which proved that the expression could be induced by low temperature. The cloning of HSP70 gene provides a stable foundation for further study of D. officinale cultivation and the breeding of the cold resistance strains.

[Effects of aridity on carbon, nitrogen, and phosphorus contents in soils and plants of mountain swamps, Zhejiang, China]. / 旱化对浙江山地沼泽湿地土壤与植物碳氮磷含量的影响.

Mountain swamps in Zhejiang Province have been suffered from severe drouhgt threats because of climate change and artificial interruption. Sphagnum bogs and swamps were gradually degraded into arid swamps. However, the effects of arid processes on the C, N, P contents of soils and their stoichiometry in mountain swamps are still unclear. We measured C, N and P concentrations, pH values, and bulk density in the upper 0-60 cm soil layers in the stands of five mountain swamps with the different arid levels. Moreover, the aboveground biomass and the C, N, P concentrations in the crushed plant mixture were also measured. The results showed that the soils of mountain swamps in Zhejiang Province were rich in soil organic carbon (SOC), total nitrogen (TN), but infertile in phosphorus (TP). Aboveground biomass, soil pH, bulk density increased, while SOC, TN, TP, C:N, C:P, N:P decreased with aridity. Soil pH and bulk density had significant negative correlations with SOC, TN, and TP in soils, respectively. The differences in the C, N, P accumulation in the soils were probably associated with litterfall production, the oxygen condition of wetlands, and the degree of plant decomposition at the different types of mountain swamps. In all, arid trends were obvious at the mountain swamps in Zhejiang Province. Soil nutrients, such as C, N, P, deceased, while plant community succeeded from the wet swamp to the mesophyte vegetation with the arid processes. The contents of C, N and P in the plants varied across species, and were not coupled with those in the soils.

Plasma Exosomal miRNA-122-5p and miR-300-3p as Potential Markers for Transient Ischaemic Attack in Rats.

Background: Differentiation of transient ischaemic attack (TIA) from ischaemic stroke within the thrombolysis time window is difficult. Although TIA may be diagnosed within this window, the latest imaging technologies are complex and costly. Serum markers, which are non-invasive, rapid and economic, are used for diagnosis and prognosis of various diseases. Exosome-derived miRNA markers for TIA are unknown. Methods: We examined focal brain ischaemia produced by occlusion of the middle cerebral artery (MCAo) for 5 min, 10 min, and 2 h in rats. Exosomal miRNAs with consistent trends in cerebrospinal fluid (CSF) and plasma were identified by deep sequencing and quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic (ROC) curve were used to evaluate the diagnostic accuracy of these miRNAs for TIA in rats. Results: Rno-miR-122-5p and rno-miR-300-3p were selected. Plasma exosomal rno-miR-122-5p was significantly downregulated in 10 min ischaemic rats compared with control and 5 min plasma. Plasma exosomal rno-miR-300-3p was significantly upregulated in 5 min ischaemic rats compared with control, 10 min and 2 h rats. Plasma and CSF levels of these miRNAs were correlated. ROC analysis showed high AUC values for rno-miR-122-5p (0.960) and rno-miR-300-3p (0.970) in the 10 and 5 min rats, respectively, compared with controls. Conclusions: Plasma exosomal rno-miR-122-5p and rno-miR-300-3p may be blood-based TIA biomarkers.

Plasma Exosomal miR-422a and miR-125b-2-3p Serve as Biomarkers for Ischemic Stroke.

BACKGROUND: MircroRNA (MiRNA) levels are associated with disease pathophysiology and are high in plasma exosomes. Plasma exosomal miRNAs serve as potential therapeutic targets and diagnosis biomarkers in some diseases but few studies have examined them in Ischemic Stroke (IS). Therefore, we explored the potential predictive value of plasma exosomal miR-422a and miR-125b-2-3p in different IS phases (acute and subacute phases). METHODS: Fifty-five IS patients and 25 age and sex matched healthy controls were recruited. Patients were classified into two groups: 27 patients in acute phase (days 1-3) and 28 patients in subacute phase (days 4-14). The plasma exosomal levels of miR-422a and miR-125b-2-3p were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The Areas Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve were constructed to evaluate the diagnostic accuracy of these miRNAs in IS. RESULTS: The expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group (P<0.001, P<0.001, respectively), and the miR-422a levels were increased in the acute phase group (P<0.005) as compared to the controls. Additionally, the expression levels of plasma exosomal miR-422a and miR-125b-2-3p were significantly decreased in the subacute phase group than in the acute phase group (P<0.001, P<0.005, respectively). ROC analysis showed high AUC values for miR-422a and miR-125b-2-3p in the subacute phase group as compared to those in healthy controls: 0.971 and 0.889, respectively, and miR-422a in the acute phase group as compared to healthy controls were 0.769. CONCLUSION: Plasma exosomal miR-422a and miR-125b-2-3p may serve as blood-based biomarkers for monitoring and diagnosing in IS patients, with plasma exosomal miR-422a showing the best diagnostic value. The use of these two plasma exosomal miRNAs in combination may be powerful for determining IS stage.

[Experimental study of individualized cancer immunotherapy based on dendritic cells against gastric cancer].

OBJECTIVE: To investigate the antitumor effects of cytotoxic T lymphocytes (CTLs) induced by autologous dendritic cells that were inspired by autologous tumor lysates (ATLs-mDCs). METHODS: Primary gastric cancer cells prepared by short-term culture were used as targets. ATLs-mDCs were subjected to activate autologous T cells to generate CTLs. The immunological functions of DCs were evaluated by flow cytometry and by mixed leukocyte response (MLR) assay. The antitumor outcome of tumor antigen specific CTLs was tested by cytotoxicity assay. Concentrations of IL-12 in cultured DCs and INF-gamma in CTLs were measured by ELISA. RESULTS: The expressions of MHC-II, CD80, CD83 and CD86 were significantly up-regulated in ATLs-mDCs, moreover, the ATLs-mDCs obtained the capability of stimulating the proliferation of autologous T cells with high efficiency. The secretion of IL-12 in ATLs-mDCs was significantly higher than that in pure mature DCs (t = 15.47, P < 0.01) and in immature DCs (t = 28.44, P < 0.01). The secretion of INF-gamma in CTLs activated by ATLs-mDCs was significantly higher than that in CTLs by pure mature DCs (t = 4.84, P < 0.05) and in CTLs by immature DCs (t = 13.74, P < 0. 01). The antigen specific cytotoxicity of CTLs induced by ATLs-mDCs was significantly higher against autologous tumor cells [(84 +/- 11)%] than that against two allogeneic tumor cell lines [(19 +/- 7)% and (19 +/- 11)%; t = 54.18 and 56.46, P < 0.01, respectively]. CONCLUSIONS: ATLs-mDCs might mediate the antigen specific CTLs against autologous gastric cancer cells ex vivo with high efficiency.

[Effect of land consolidation on soil microbial community diversity.] / åœŸåœ°æ•´ç†å¯¹åœŸå£¤å¾®ç”Ÿç‰©ç¾¤è½å¤šæ ·æ€§çš„å½±å“.

Land consolidation has become a main means of achieving the intensive use of land resources, which ensures the dynamic equilibrium and requisition-compensation balance of the total cultivated land in China. However, the intensive disturbance during the land consolidation may affect the soil quality. In order to investigate the effect of land consolidation on soil microbial diversity, we studied the changes of soil microbial community diversity after 1-year and 4-year land conso-lidation by using PLFA method. The results indicated that compared with no consolidation (Z0), for land consolidation after 1a (Z1a), the soil pH value increased by 14.6%, the soil organic carbon content decreased by 65.4%, the phospholipid fatty acids content and relative abundance of all the microflora decreased significantly (P<0.05) by 43.4%-63.7% and 25.2%-53.9%, respectively, and the ratio of fungi/bacteria (F/B) decreased significantly by 35.9% (P<0.05), while the ratio of Gram-positive bacteria/Gram-negative bacteria (G+/G-) increased significantly by 56.1%. These were significantly related to the increased pH value and the decrease of organic carbon content. The Shannon index and evenness index (E) of soil microbial diversity were significantly decreased, with significant differences observed among Z0 and Z1a, Z4a. After 4-year land consolidation, the indices characterizing soil microbial community diversity were improved compared with those after 1-year land consolidation. In summary, the land consolidation could significantly affect the composition of soil microbial communities, and decrease the stability of the soil ecosystem.